The invention relates to novel modified oligonucleotides, the construction thereof, and their use in oligonucleotide-based therapies. More specifically, the invention is to novel oligonucleotides having modified internucleoside linkages which are resistant to nucleases, having enhanced ability to penetrate cells, and which are capable of binding target oligonucleotide sequences in vitro and in vivo. The modified oligonucleotides of the invention are particularly useful in oligonucleotide-based therapies utilizing the modified oligonucleotides to interrupt protein synthesis or otherwise inactivate messenger RNA or double stranded DNA.
The application of oligonucleotides and oligonucleotide analogs (oligomers) for therapeutic uses represents a relatively new development in drug design and discovery. Several fundamental therapeutic approaches that utilize oligomers have been proposed.
One approach is based largely on interfering with gene expression through oligomer binding to a complementary RNA sequence. This application is known as xe2x80x9cantisensexe2x80x9d therapy because the oligomer base sequence is identical to the antisense strand of the gene that gave rise to the RNA (Uhlmann, E., et al., Chem Reviews (1990) 90:543-584;and Stein, C. A., et al., Cancer Res (1988) 48:2659-2668). Another approach, referred to herein as xe2x80x9ctriple helixxe2x80x9d therapy utilizes oligomers that bind to duplex DNA as detailed below. Binding to a target DNA is sequence specific but involves different base pairing binding. Both antisense and triple helix therapies exert therapeutic effects via binding to nucleic acid sequences that are responsible for disease conditions. Such sequences are found in the genome of pathogenic organisms including bacteria, protozoa, yeasts, parasites, fungi or viruses or may be endogenous sequences (oncogenes). By modulating the expression of a gene important for establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
Another therapeutic approach that is based on the use of oligomers includes generation of xe2x80x9captamersxe2x80x9d and is disclosed and claimed in commonly owned application Ser. Nos. 745,215, 659,980 and 658,849.This approach utilizes oligomers that specifically bind to proteins thereby interfering with their function. The use of oligomers that mimic the structure of certain RNA molecules that are bound by intracellular proteins has also been adduced as a therapeutic approach as described in international application no. PCT/US91/01822.
Antisense oligonucleotides are synthetic oligonucleotides which bind complementary nucleic acids (i.e. sense strand sequences) via hydrogen bonding, thereby inhibiting translation of these sequences. Therapeutic intervention at the nucleic acid level using antisense oligonucleotides offers a number of advantages. For example, gene expression can be inhibited using antisense or triple helix oligomers. Inhibition of gene expression is more efficient than inhibition of the protein encoded by the gene since transcription of a single DNA sequence gives rise to multiple copies of mRNA which, in turn, are translated into many protein molecules.
Antisense and triple helix therapies for diseases whose etiology is characterized by, or associated with, specific DNA or RNA sequences, are particularly useful. The oligomer employed as the therapeutic agent can be directly administered or generated in situ and is one that is complementary to a DNA or RNA needed for the progress of the disease. The oligomer specifically binds to this target nucleic acid sequence, thus disturbing its ordinary function.
An oligomer having a base sequence complementary to that of an mRNA which encodes a protein necessary for the progress of the disease, is particularly useful. By hybridizing specifically to this mRNA, the synthesis of the protein will be interrupted. However, it is also possible to bind double-stranded DNA using an appropriate oligomer capable of effecting the formation of a specific triple helix by inserting the administered oligomer into the major groove of the double-helical DNA. The resulting triple helix structure can then interfere with transcription of the target gene (Young, S. L. et al., Proc Natl Acad Sci (1991) 88:10023-10026).
An important feature of therapeutic oligomers is the design of the backbone of the administered oligomer. Specifically, the backbone should contain internucleoside linkages that are stable in vivo and should be structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage. At the same time, the oligomer must also retain its ability to hybridize to the target DNA or RNA. (Agarwal, K. L. et al., Nucleic Acids Res (1979) 6:3009; Agarwal, S. et al., Proc Natl Acad Sci USA (1988) 85:7079.) In order to ensure these properties, a number of modified oligonucleotides have been constructed which contain alternate internucleoside linkages. Several of these oligonucleotides are described in Uhlmann, E. and Peyman, A., Chemical Reviews (1990) 90:543-584. Among these are methylphosphonates (wherein one of the phosphorous-linked oxygens has been replaced by methyl); phosphorothioates (wherein sulphur replaces one of these oxygens) and various amidates (wherein NH2 or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen). These substitutions confer enhanced stability, for the most part, but suffer from the drawback that they result in a chiral phosphorous in the linkage, thus leading to the formation of 2n diastereomers where n is the number of modified diester linkages in the oligomer. The presence of these multiple diastereomers considerably weakens the capability of the modified oligonucleotide to hybridize to target sequences. Some of these substitutions also retain the ability to support a negative charge and the presence of charged groups decreases the ability of the compounds to penetrate cell membranes. There are numerous other disadvantages associated with these modified linkages, depending on the precise nature of the linkage.
It has also been suggested to use carbonate diesters. However, these are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorous in the phosphodiester. Similarly, carbamate linkages, while chiral, confer trigonal symmetry and it has been shown that poly dT having this linkage does not hybridize strongly with poly dA (Coull, J. M., et al., Tet Lett (1987) 28:745; Stirchak, E. P., et al., J Org Chem (1987) 52:4202.
WO 91/15500, published Oct. 17, 1991, teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage typically sulfamate diesters which are isosteric and isoelectric with the phosphodiester.
WO 89/12060, published Dec. 14, 1989, similarly discloses linkages containing sulfides, sulfoxides, and sulfones.
WO 86/05518, published Sep. 25, 1986, suggests a variant of stereoregular polymeric 3xe2x80x2,5xe2x80x2 linkages.
U.S. Pat. No. 5,079,151 to Lampson et al., discloses a msDNA molecule of branched RNA linked to a single strand DNA via a 2xe2x80x2,5xe2x80x2 phosphodiester linkage.
Commonly owned, pending U.S. patent application Ser. No. 690,786, filed Apr. 24, 1991, the entirety of which is incorporated by notice, describes modified linkages of the formula xe2x80x94Yxe2x80x2CXxe2x80x22Yxe2x80x2xe2x80x94 wherein Yxe2x80x2 is independently O or S and wherein each Xxe2x80x2 is a stabilizing substituent and independently chosen. Amide-containing linkages disclosed in commonly owned, pending U.S. patent application Ser. No. 07/889,736, filed May 28, 1992, S. Swaminathan, et al inventors, the entirety of which application is incorporated herein by reference, are also suitable for incorporation into oligomers containing one or more of the linkages disclosed herein.
Modifications of oligomers that enhance their affinity for target molecules will generally improve the therapeutic potential for those compounds. Previous approaches to improve binding affinity for complementary nucleic acids have centered primarily on (i) covalent linkage of intercalating agents to oligomers (Asseline, U., et al., Proc Natl Acad Sci (1984) 81:3297-3401), (ii) introduction of modified bases to form more stable base pairs (Inoue, H. et al., Nucl Acids Res (1985) 13:7119) and (iii) altering the charge characteristics of oligomer internucleotide linkages (Letsinger, R. L. et al., J Am Chem Soc (1988) 110:4470). Morpholino-type internucleotide linkages are described in U.S. Pat. No. 5,034,506 and in some cases give rise to an increased affinity of the oligomer for complementary target sequences.
Commonly owned pending U.S. patent applications Ser. No. 07/763,130, filed Sep. 20, 1991, and Ser. No. 07/690,786 disclose modified oligonucleotides having modified nucleoside linkages which are capable of strong hybridization to target RNA and DNA.
That disclosure suggests that 3xe2x80x2,5xe2x80x2 internucleoside linkages are especially appropriate since the resulting oligonucleotides are stable in vivo, resistant to endogenous nucleases, and are able to hybridize to target nucleotide sequences. The structure of the oligonucleotide backbone suggests that 3xe2x80x2,5xe2x80x2 linkage is optimal for a phosphodiester linkage in view of the steric size of the phosphodiester group and the related bond lengths, angles, and torsions involved. Although a phosphodiester should be suitable for the 2xe2x80x2,5xe2x80x2 linkage since the O2xe2x80x2xe2x80x94P, Pxe2x80x94O5xe2x80x2 bond lengths, angles, and torsions are all appropriate, the steric bulk of the phosphodiester is too large for the space available between the 5xe2x80x2 sugar (2xe2x80x2 and 3xe2x80x2 atoms) and the 3xe2x80x2 sugar (O4xe2x80x2 and C5xe2x80x2). Our discovery of the 2xe2x80x2,5xe2x80x2 linkage and its characteristics is based on modeling studies that both (i) predicted such linkages in a binding-competent oligonucleotide and (ii) defined the range of molecular characteristics such linkages could assume without loss of binding competence. Binding competence, as used herein, refers to either Watson-Crick pairing with single-stranded DNA or single-stranded RNA or to Hoogsteen pairing (Beal, P. A. et al., Science (1991) 251:1360-1363) with duplex nucleic acids including duplex DNA or duplex RNA.
Although space available for the 2xe2x80x2,5xe2x80x2 linkage has more stringent steric restraints upon it than does the space for the 3xe2x80x2,5xe2x80x2 linkage, there are fewer restrictions with regard to bond lengths, angles and the like. Because of this, the 2xe2x80x2,5xe2x80x2 linkage is fundamentally different from the 3xe2x80x2,5xe2x80x2 linkage. This restraint has been experimentally verified in that 2xe2x80x2,5xe2x80x2 phosphodiesters have been found not to be suitable for efficient hybridization (Kierzek, R. et al., Nucl Acids Res (1992) 20:1685-1690). However, the 2xe2x80x2,5xe2x80x2 linkages of this invention do not have the steric bulk of a phosphodiester but do fall within the range of requirements for bond length, angles, and torsion placed upon a linkage in that position. Consequently, our linkages are quite suitable and, in some cases, superior to 3xe2x80x2,5xe2x80x2 phosphodiester linkages.
The therapeutic potential of oligomers is generally enhanced by modifications that increase oligomer uptake by cells or reduce the rate of metabolism by cells or serum. Such modifications include (i) reduced oligomer charge, (ii) increased stability toward nuclease activity, and (iii) increased lipophilicity of the oligomer. oligomers having modified internucleotide linkages as described in the invention exhibit sequence-specific binding to complementary single stranded and duplex target sequences.
The present invention provides an internucleoside linkage which is resistant to nuclease digestion, and which is stable under physiological conditions, and which can be neutral or positively charged so as to enhance cell permeation. U.S. patent application Ser. No. 07/868,487 filed Apr. 14, 1992, the entire disclosure of which is incorporated herein by reference, describes modified oligomers that efficiently enter cytoplasm by passive diffusion. The linkages described herein are generally compatible with such permeation competent oligomers. Both nuclease stability and enhanced cellular permeation are important considerations for the development of oligonucleotide analogs that are intended to be used as therapeutic agents that function by binding to specific DNA or RNA (mRNA, hnRNA, etc.) sequences. Such specific target sequence binding underlies their therapeutic efficacy by interfering with the normal biological function of nucleic acid sequences associated with pathological conditions. Furthermore, the linkages can be achiral and thus do not lead to the problem of multiple diastereomers in the resulting compounds.
The present invention is based on the construction of novel oligonucleotides containing modified backbone linkages which linkages are also referred to as modified internucleoside linkages. These oligonucleotides are stable in vivo, resistant to endogenous nucleases and are able to hybridize to target nucleotide sequences.
In one embodiment, the present invention is directed to a modified oligonucleotide or derivative thereof, wherein the modification comprises substitution, for one or more phosphodiester linkages between 2xe2x80x2 and 5xe2x80x2 position adjacent nucleosides, with a two to four atom long internucleoside linkage wherein at least one of the atoms making up the internucleoside linkage is selected from nitrogen, oxygen and sulfur, with the remainder being carbon.
In another embodiment, the subject invention is directed to an oligomer of the formula:
(W,Y)xe2x80x94Qxe2x80x94(Zxe2x80x94Q)nxe2x80x94(W,Y)
or a derivative thereof, where each 
where X is S, O, CH2, CHF or CF2;
R1 independently is xe2x80x94O-alkyl (C1-C12), xe2x80x94S-alkyl (C1-C12), H, OH, OCH3, SCH3, OC3H5 (O-allyl), OC3C7(O-propyl), SC3H5, or F and
where R1 is on a terminal group of the oligomer, R1 may additionally be PO3xe2x88x922 or a blocking group selected from a dimethoxytrityl (DMT) moiety, a monomethoxytrityl (MMT) moiety, H-phosphonate (OPO2H), methylphosphonate (OPO2CH3) or phosphoramidite;
B is independently a purine or pyrimidine residue or an analogous residue, and
Q is independently
a phosphodiester analog or
a two to four atom long internucleoside linkage
wherein at least one of the atoms making up the internucleoside linkage is selected from nitrogen, oxygen or sulfur, with the remainder being carbon; but no two adjacent atoms are oxygen and, for three atom linkages, no 3 adjacent atoms are all nitrogen or all oxygen or all sulfur and
n is 1-100, preferably 2-28,
but where each Q and each Z in each mer (n) is independently selected.
The designation (W, Y) means that either W or Y is linked to Q at the indicated positions. In preferred embodiments, a maximum of 20% of the linkages give rise to inversion of oligomer polarity. Linkages that invert polarity occur when there is a 5xe2x80x2 to 5xe2x80x2,3xe2x80x2 to 2xe2x80x2 or 3xe2x80x2, or 2xe2x80x2 to 3xe2x80x2 or 2xe2x80x2 linkage between adjacent nucleoside residues. The preferred linkage type for the majority of linkages in most oligomers is thus 3xe2x80x2 or 2xe2x80x2 to 5xe2x80x2. When X is CH2 or CHF, the material may be produced according to published procedures (Otvos, L. et al. Tet Letters (1987) 28:6381-6384; Divakar, K. L. et al J. Chem Soc Perkin Trans I (1982) 1625; J. Chem Soc., Perk. Trans. I (1991) 2373-2377).
In preferred embodiments structure VII is included in oligomers as a nucleoside linked to an adjacent nucleoside through a riboacetal linkage. In other embodiments where VII is not linked via a riboacetal linkage, circular or branched oligomers may be obtained which are useful in oligonucleotide based therapies or diagnosis applications (Kool, E. T., J Am Chem Soc (1991) 113:6265-6266).
Oligomers are conveniently produced from dimers or trimers as synthons for solid phase or solution phase synthesis using standard methods known in the art.
In yet other embodiments, the invention is directed to methods for treating diseases mediated by the presence of a nucleotide sequence which comprise administering to a subject in need of such treatment an amount of the above modified oligonucleotides capable of specifically binding the nucleotide sequence effective so to inactivate the nucleotide sequence.
A feature of linkages wherein oxygen is located at the 2xe2x80x2 position is enhanced stability of the glycosidic bond with the heterocycle. Such linkages are included in the group of preferred linkages described in detail below.
It has been found that oligomers containing these internucleotide linkages efficiently bind complementary single-stranded and double-stranded nucleic acid sequences. Triple helix structures were formed under physiological salt conditions. Unmodified control oligomers were less efficient at forming triple helices under the same conditions. Thus, oligomers of the present invention are generally characterized as containing one or more 2xe2x80x2,5xe2x80x2 or related linkages. The analogs may be utilized in oligomers that contain additional modifications of other nucleotides that comprise the oligomer. An exemplary list of such modifications include oligomers where (i) one or more nucleotide residue is modified at the 2xe2x80x2 position, (ii) one or more crosslinking moieties have been incorporated, (iii) switchback linkers have been incorporated as described in copending U.S. application Ser. No. 07/559,958, incorporated herein by reference, (iv) other substitute internucleotide linkages have been included and (v) base analogs that facilitate duplex or triplex formation, such as 8-oxo-N6-methyladenine, 5-propynyluracil, 5-propynylcytosine or 7-deazaxanthine have been included. One or more of such modifications may advantageously be incorporated into a given oligomer depending upon target nucleic acid sequences.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.